One of the paramount goals of medical care is the development of modern vaccines for prophylaxis and efficient delivery of therapeutic substances for the treatment of diseases. So far, virosomes are known as suitable vesicles for antigen-delivery and/or as carrier for therapeutic substances.
Virosomes are complexes composed of lipids and at least one viral envelope protein, produced by an in vitro procedure. The lipids are either purified from eggs or plants or produced synthetically, and a fraction of the lipids originates from the virus providing the envelope protein. Essentially, virosomes represent reconstituted, empty virus envelopes devoid of the nucleocapsid including the genetic material of the source virus(es). Virosomes are not able to replicate but are pure fusion-active vesicles. These virosomes are functional in that their membrane fusion activity closely mimics the well defined low-pH-dependent membrane fusion activity of the intact virus, which is solely mediated by the viral fusion protein. Like viruses, virosomes are rapidly internalized by receptor-mediated endocytosis or fusion with the cell membrane.
Mostly, the virosomes utilized are virosomes termed immunopotentiating reconstituted influenza virosomes (IRIVs). IRIVs are spherical, unilamellar vesicles with a mean diameter of 150 nm and comprise a double lipid membrane, consisting essentially of phospholipids, preferably phosphatidylcholines (PC) and phosphatidylethanolamines (PE). IRIVs contain the functional viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) intercalated in the phospholipid bilayer membrane. The biologically active HA does not only confer structural stability and homogeneity to virosomal formulations but also significantly contributes to the immunological properties by maintaining the fusion activity of a virus. Optionally, the IRIVs comprise hemagglutinin molecules of more than one virus strains, thus forming chimeric IRIVs.
IRIVs have been developed by incorporating the hemagglutinin (HA) from an influenza A strain into liposomes composed of phosphatidylcholin. The influenza virus surface glycoprotein HA guides the virosomes specifically to antigen-presenting cells and leads to fusion with their endosomal membrane. This process provides optimal processing and presentation of the antigens to immunocompetent cells. The T lymphocytes are activated to produce cytokines which in turn stimulate the B lymphocytes to form large amounts of specific antibodies. Moreover, the stimulation of B lymphocytes also occurs through direct contact with the antigen-virosome complex.
Virosomes are highly effective adjuvant/carrier systems in modern vaccination/therapy, possessing superior properties as antigen delivery vesicles and a strong immunogenic potential whilst concomitantly minimizing the risk of side effects. Moreover, virosomes show adjuvant (WO92/19267), trans-adjuvant (European patent application EP05027624) and a non-specific immune stimulating effect (European patent application EP06027120).
For more than 50 years, influenza vaccines have been produced in embryonated chicken eggs. However, the conventional standard methodology is extremely lengthy and cumbersome. Current egg-derived vaccine production requires up to nine months from the isolation of a newly identified virus strain to the final product. This may hinder the response to unanticipated demands such as the discovery of pandemic strains, production failures and seasonal influenza virus strain changes. Moreover, the traditional egg-based methodology requires a huge amount of eggs, an adaptation of the virus isolate to the egg and an extensive purification to reduce the amount of contaminating egg proteins and to minimize the risk of allergies against egg albumins.
In contrast, a cell line-based process is faster and more flexible with respect to virus propagation and allows the production of strains that cannot be adequately grown in eggs (e.g. Avian Hong Kong Flu in 1997). Moreover, the use of cell lines for manufacture of viruses has several advantages in connection with the safety of the resulting vaccine: no antibiotic additives are present in the vaccine formulation; no toxic preservatives (such as thiomersal) are needed; endotoxin levels are reduced, no egg allergy may be caused; growth takes place in protein and serum free media (no adventitious agent/BSE); the virus vaccine preparations are of high purity.
Recently, there have been considerable efforts to develop cell culture systems for vaccine production. Most of the known cell culture systems are based on mammalian cell lines such as e.g. Vero cells, MDCK cells, BHK cells and PerC6 cells. There have been a number of reports on vaccine development based on mammalian cell culture systems. However, virus vaccines produced in said mammalian cell culture systems suffer from the risk of autoimmune reactions to mammalian cell-derived proteins.
The virosome fusion process is essential for an efficient antigen/drug delivery (Schoen P, et al., 1999). Therefore, there is a need in the art to develop virosomes with improved quality with respect to their fusogenic activity and immunogenicity.